Very often a primary goal in making measurements of substances in biological fluids is to provide information about some enduring state of the organism which produces those fluids, such as when biochemical measures are taken to provide information for the diagnosis of disease.
It often happens that the concentrations of these substances in the biological fluids are subject to temporal fluctuations, such that a series of samples taken from the biological fluids would reveal a distribution of concentration values. Generally it is not the concentration present at a given instant, but rather the extended summation of the fluctuating concentrations over some time interval, which is of ultimate interest. This summation of instantaneous levels over a defined time interval, in mathematical terms, would be called the "time-integral".
The time-integral is preferred because it is more representative of the enduring state of the organism than are the temporally-fluctuating instantaneous values. The time integral, divided by the length of the time interval over which it was obtained, is equivalent to the true average concentration of the substance over that interval.
This advantage of the time-integral over instantaneous values is illustrated by Goldzieher et al (Journal of Clinical Endocrinology and Metabolism 43:824 (1976)). These authorities estimated the 8-hour mean of blood plasma luteinizing hormone concentrations in normal men by a prior art method (described below), then calculated the probability that a single plasma sample would yield a value within plus or minus 20 percent of this mean.
They estimated this probability was only 30%, i.e., that a single plasma measurement was a very poor indicator of the enduring state of the organism, with regard to this important substance.
The practical diagnostic advantage of time-integrated measurement can be illustrated by another example from the medical literature. Zadik et al (Journal of Clinical Endocrinology and Metabolism 51:1099, (1980)) compared the ability of three clinical tests to distinguish among three classes, i.e., normal persons, patients with mild essential hypertension, and patients with Cushing's syndrome. Three tests were used for each of those three classes of patients: (1) a prior-art method (described below) of estimating the time integral of plasma cortisol, (2) measurement of urinary free cortisol, and (3) measurement of urinary 17-hydroxycorticosteroids. The latter two methods are recognized to be far superior to instantaneous plasma cortisol measurement, and are considered to be the standard measures in current medical practice.
However, the time-integrated plasma cortisol measurement was found to be clearly superior to the standard measures in discriminating among these groups. The authors nevertheless declined to recommend the time-integrated measurement as a new standard of practice, however, because the prior art heretofore available for that improved method is exceedingly complex.
It often happens that compounds of biological interest (e.g., hormones, drugs, amino acids, etc.) exist in biological fluids in two states or fractions, i.e., those molecules which are in association with macromolecules (generally proteins) such that their movements and reactions are in some way limited by the macromolecules, and those which are free of such association.
The latter fraction is often called the "free" fraction and the former the "bound" fraction; and in general, the proportion of the bound and free fractions is determined by the concentrations of the various biochemical components of the biological fluid, by temperature, and by other factors of a physical or chemical nature.
When the compounds of interest exist in both "bound" and "free" states, it is often observed that the biological effects of the compounds seem to be exerted only by the free fraction (see, for example, Lasnitzki and Franklin, Journal of Endocrinology 64:289, (1975)).
Indeed, it is found that the free fraction concentration of a hormone in a biological fluid is a much better indicator of the state of the organism than is the total concentration. Vermeulen et al (Journal of Endocrinology and Metabolism 33:758, (1971) present many cases illustrating the superiority of measurements of free testosterone concentration obtained by a prior art method (described below), over simple measurement of the total hormone concentration, again in the context of medical diagnosis.
Essentially, the prior art as to the measurement of the time-integral of substances in biological fluids consists of two similar means and methods. As now described, they are seen to both fail to achieve the advantages of the present invention, and, instead, both require the removal of samples or specimens over a prolonged time period, and other differences as noted herein.
The means and method described by Kowarski et al (Journal Clinical Endocrinology and Metabolism 32:356, (1971)) is typical of those using a prolonged sample-removal procedure, this type being a constant-withdrawal system (U.S. Pat. No. 3,908,657) to obtain a sample.
In the Kowarski system, a mechanical pump is used to obtain a sample of the biological fluid at a constant rate over a long time interval. The content of the relevant substance in the sample is then measured by suitable means; and since each instant of the elapsed interval is represented by an equal amount of fluid in the sample, the concentration of the compound of interest in the whole sample directly reflects the time integral of its concentration in the biological fluid over the pumping interval. This is similar to the method employed by Zadik et al (1980).
In practice, this system has several disadvantages, relative to the invention to be described here, and the disadvantages are now summarized.
First, the Kowarski system involves the use of a miniaturized precision pump which is very expensive to manufacture, relative to the device of the invention to be described.
Second, a very bothersome disadvantage is that a specially-treated catheter must be inserted in a major blood vessel of the subject, and maintained without thrombosis for the duration of the measurement procedure; this requirement makes the practice of the Kowarski invention stressful for some if not most subjects and even impossible for others (such as some animals which will not tolerate the externalized catheter connection).
Third, the continuing sample-removal procedure of the prior art has the disadvantage that the necessity to avoid thrombosis in the catheter places limits on the slowness with which blood can be withdrawn, and places limits upon the duration of the sampling period; and these limits are so restrictive that small animals (such as laboratory rats) are not suitable subjects due to their limited blood volume, and most subjects cannot reliably be monitored for more than 24 hours without changing catheters.
Fourth, as a disadvantage, and even if the other obvious bothers and distress to the subject were not present, the subject must either be severely limited in its movements, or else must carry the pump and associated apparatus along with it by some means.
Fifth, this prior art method has been applied only to blood.
A second means and method shown in the prior art, also of prolonged sample-removal nature, is typified by the method of Goldzieher et al (1976). In that method, multiple instantaneous samples are obtained frequently and at regular intervals throughout the measurement period. The concentration of the compound of interest is measured by suitable means in each sample, and the results are then mathematically integrated to yield an estimate of the time integral.
That prior art method, because it too is of course of prolonged sample-removal type even though of repetitive rather than continuous nature, shares many disadvantages with the previously described prior art, and is generally more laborious, as will be apparent when it is considered that either a catheter must be maintained as before, or repeated blood samples otherwise drawn, and that a large number of samples of the blood plasma must be measured by suitable means, thus more disadvantageous in this respect than the Kowarski system described above in which a single pooled sample was involved.
The prior art as to measurement of the free fraction of biochemical substances consists of a number of devices for accomplishing a physical separation of the macromolecules which bind the compounds of interest, from some remaining part of the sample, without seriously disturbing the proportion of the bound and free fractions. All such prior art devices fail to provide advantages of the present invention, in that they require further procedures in addition to that of an already complex analysis.
Among the oldest methods is equilibrium dialysis, as shown in Schellman et al, Journal of the American Chemical Society 76:2808 (1954), in which is used a membrane which permits the passage of the free compound of interest, but not the macromolecules and the associated bound compound. The membrane is arranged so that it serves as a differentially-permeable barrier between the sample with its bound and free fractions, and a small volume of a similar fluid, which contains neither the macromolecules nor the bound or free portions of the compound of interest.
With this type of device, substances which can pass through the membrane do so in accordance with the laws of diffusion; and eventually an equilibrium is reached wherein the concentration of the "free" compound of interest is equal on both sides of the membrane, while the macromolecules on the "sample" side of the membrane continue to sequester the "bound" fraction.
The small volume containing only "free" compound is then removed and the total concentration therein measured by suitable means, thus yielding an estimate of "free" compound concentration in the original sample.
Other methods in the prior art accomplish the separation of macromolecules by causing them to be insoluble in the fluid without causing them to release the "bound" molecules. The means for making the macromolecules insoluble differ from one case to another, but generally involve the introduction of chemical species which combine with the macromolecules to produce an insoluble complex, which is an additional step not required by the present invention.
Perhaps the advantages and significant nature of the inventive concepts set forth herein may be realized by reference to recent advances in related art, which yet fail to accomplish the achievements in this invention. Cheesman et al (Fertility and Sterility 38:475, (1982)) have reported a method whereby a binding preparation is implanted in an animal, for the purpose of binding and sequestering a circulating hormone and depleting its concentration throughout the blood plasma of the animal. This art differs from the present invention in that the pathway, by which the hormone has access to the binding protein, has unknown and non-constant permittivity, so that no useful measurement can be obtained thereby.
Further, this related art is designed expressly to deplete and change the level of the hormone, and thereby to preclude useful measurement.
This present invention, quite in contrast, avoids depletion of, and allows measurement of, the time integrals of compounds such as hormones, by virtue of a pathway of precisely known constant, and limited permittivity.
Similarly, Goode (British Journal of Pharmacology 41:558, (1971)) has described a device somewhat superficially similar to that preferred in the present embodiment of this invention, but the Goode device is neither designed for, nor suitable for use, in the practice of this invention. The Goode device incorporates a tubular reservoir, which communicates with its immediate environment through a single port of dialysis membrane installed at one end of the tubular reservoir, while at the other end two small tubes are fitted. Further, the Goode device is a subcutaneously-implanted reservoir for the chronic delivery of drugs, which operates by the diffusion of drugs from the reservoir, through the membrane, and into the surrounding environment. It is provided with externalized tubes for the purpose of replenishing the drugs contained in the reservoir.